The accomplishment from the two main projects of the unit are: 1) Development of a robust protein recombinant expression and purification methodology to test salivary proteins for biological activities, and validate vaccine candidates identified using DNA immunization. We maximized expression of a properly folded recombinant salivary protein by cloning sand fly salivary gland transcripts into the mammalian expression vector developed by this unit, VR2001-TOPO, the same vector used for DNA immunization, and used it to transfect 293F mammalian cells. Transfected cells produced soluble recombinant proteins and by following an optimized purification methodology we obtained a number of salivary proteins in large quantities, highly pure and with a minimal amount of endotoxin for testing in vaccine, immunological and biological assays. 2) The demonstration that a recombinant sand fly salivary protein protects against challenge with L. major infected sand flies. We demonstrated that the protection observed by immunization with DNA vaccine is not a bystander effect of the DNA plasmid and it is specific to the protein encoded by the plasmid. We tested the protective effect of recombinant protein LJM11 that is encoded in the selected DNA plasmid against the virulent challenge of L. major infected Lu. longipalpis sand fly bites. Mice immunized with a small amount of recombinant LJM11 in the absence of adjuvants were protected against challenge by bites from ten infected sand flies (Figure 4). This is the first demonstration of the protective role of a recombinant sand fly salivary protein against cutaneous leishmaniasis and validates the use of DNA immunization for protection against Leishmania infections. 3)Identification of two potential salivary vaccine candidates for the prevention of canine visceral leishmaniasis using a reverse antigen screening (RAS) approach. Dogs are the main reservoir of visceral leishmaniasis in Europe and South America. We developed an experimental model that mimics the natural exposure of dogs to sand fly bites and explored the immune responses to salivary proteins from the sand fly Lutzomyia longipalpis, the vector of visceral leishmaniasis in South America. Lu. longipalpis salivary proteins induced a DTH response at the bite site in the skin of dogs following repeated exposures to sand flies. This skin response is a surrogate of cellular immune response and a first screen marker of protection against Leishmania infection in rodent models. To identify the salivary components producing a cellular immune response (DTH response), specifically a protective immune response in dogs, we performed a reverse antigen screening procedure by single intradermal injection of 35 different DNA plasmids in dogs previously exposed to sand flies. This screening led to the identification of six DTH inducing salivary antigens. We further validated these results with recombinant proteins expressed in 293 F cells resulting in the confirmation of two DTH inducing salivary proteins, LL1 and LL3. We have previously demonstrated that a DTH response alone to a salivary protein is not sufficient to predict protection against Leishmania infection and that the presence of IFN-gamma as part of the DTH response is a better correlate of protection. We therefore, tested the immune profile of these two salivary vaccine candidates and both of these salivary molecules produced a DTH response with the presence of INF-gamma. We propose that the identified salivary antigens, LL1 and LL3 have the potential to be part of a future L. infantum chagasi canine vaccine based on vector antigens. 4) Identification of biological activities from novel salivary molecules of unknown sequence or function. Most sand fly salivary proteins, including the identified protective molecules, are novel and do not have an assigned biological function. Soluble recombinant proteins were produced in a mammalian expression system to test potential biological activities. We identified a potent anticoagulant salivary protein from Lu. longipalpis that binds specifically the active site of factor Xa. We also identified four salivary recombinant proteins that inhibited the alternate pathway of complement by binding specifically to C3b and one protein that inhibits the classical pathway of complement. These molecules have the potential for use in the treatment of a variety of human ailments. This work not only demonstrates that the anticoagulant inhibitor of sand flies is a novel prothrombinase inhibitor, but also makes it a potential active drug to correct pro-coagulant disorders in human medicine. C3b-inhibitors could be potential new drugs that correct human diseases related to complement such as auto-immune diseases and post-surgery complications due to complement.